Sulfated fucan oligosaccharide

ABSTRACT

A smaller molecule obtainable by allowing a sulfated fucan-digesting enzyme which digests a novel sulfated polysaccharide derived from an alga belonging to Laminariales to act on a sulfated fucan, and a method for producing the same.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a sulfated fucan oligosaccharidewhich is useful in a field of glycotechnology, and a method forproducing the same.

[0003] 2. Description of Related Art

[0004] Brown algae contain a variety of sulfated polysaccharides. Thesepolysaccharides are often generically called fucoidans or fucoidins. Inmany cases, their structures vary depending on the algae from which theyderive. For example, sulfated polysaccharides extracted from Fucusvesiculosus, Laminaria japonica, Cladosiphon okamuranus, Nemacystusdecipiens and sporophyll of Undaria pinnatifida have structuresdifferent each other. Therefore, it is necessary to obtain enzymes thatdigest the respective sulfated polysaccharides in order to obtainoligosaccharides from the sulfated polysaccharides by enzymaticallydigesting them or to determine their structures.

[0005] Molecular species of sulfated polysaccharides including sulfatedfucans, sulfated fucoglucuronomannans and sulfated fucogalactans as wellas several other molecular species have been reported. Sulfatedpolysaccharides generally have some biological activities in many cases.For example, a sulfated fucan fraction has been reported to have astrong anticoagulant activity, and a sulfated fucoglucuronomannanfraction has been reported to have an apoptosis-inducing activityagainst tumor cells.

[0006] If a sulfated polysaccharide is to be developed as apharmaceutical, it is necessary to determine its structure. It is veryadvantageous to determine the structure using an enzyme that digests thesulfated polysaccharide. However, no enzyme that digests a sulfatedpolysaccharide from a brown alga is commercially available. In addition,a digesting enzyme that specifically digests the sulfated polysaccharideof which the structure is to be determined is required. This is becausesulfated polysaccharides from brown algae vary depending on the speciesof the algae in many cases. Structures of sulfated polysaccharidesderived from algae belonging to Laminariales have been studied, althoughstructures have been revealed only for a few kinds among many molecularspecies.

[0007] As described above, a structurally homogeneous sulfated fucanoligosaccharide which is produced by enzymatic means using an enzymethat digests a novel sulfated polysaccharide derived from an algabelonging to Laminariales (i.e., an enzyme that specifically digests asulfated fucan) has been desired.

[0008] Thus, the main object of the present invention is to provide asmaller molecule obtainable by allowing a sulfated fucan-digestingenzyme which digests a novel sulfated polysaccharide derived from analga belonging to Laminariales to act on a sulfated fucan, and a methodfor producing the same.

SUMMARY OF THE INVENTION

[0009] The first aspect of the present invention relates to a sulfatedfucan having the following chemical and physical properties:

[0010] (1) containing fucose as a constituting saccharide;

[0011] (2) containing a sulfated saccharide of general formula (I) as anessential component of the constituting saccharide:

[0012] wherein R is H or SO₃H, at least one of Rs is SO₃H and n is aninteger of 1 or more; and

[0013] (3) being converted into smaller molecules by a sulfatedfucan-digesting enzyme derived from Alteromonas sp. SN-1009 to generateat least one compound selected from the group consisting of thecompounds of general formulas (II), (III), (XIII), (XIV), (XV) and(XVI):

[0014] wherein R is H or SO₃H and at least one of Rs is SO₃H in allformulas above, or a salt thereof.

[0015] The second aspect of the present invention relates to a sulfatedfucan oligosaccharide of general formula (I):

[0016] wherein R is H or SO₃H, at least one of Rs is SO₃H and n is 1 to5.

[0017] The third aspect of the present invention relates to a method forpreparing a sulfated fucan oligosaccharide, the method comprising:

[0018] allowing a sulfated fucan-digesting enzyme derived fromAlteromonas sp. SN-1009 to act on a sulfated fucan of the first orsecond aspect; and

[0019] collecting a digestion product.

[0020] According to the third aspect, the sulfated fucan is derived fromKjellmaniella crassifolia, Laminaria japonica or Lessonia nigrescens.

[0021] The fourth aspect of the present invention relates to a sulfatedfucan oligosaccharide obtainable by the preparation method of the thirdaspect.

[0022] The fifth aspect of the present invention relates to a sulfatedfucan oligosaccharide having a chemical structure selected from thegroup consisting of general formulas (II), (III), (XIII), (XIV), (XV)and (XVI):

[0023] wherein R is H or SO₃H and at least one of Rs is SO₃H in allformulas above, or a salt thereof.

[0024] As a result of intensive study, the present inventors have founda method for producing a sulfated fucan oligosaccharide by digesting anovel sulfated polysaccharide derived from a alga belonging toLaminariales using a sulfated fucan-digesting enzyme. The sulfated fucanoligosaccharide can be utilized as a reagent for glycotechnology and isstructurally homogeneous. Furthermore, the present inventors havedetermined the structure of the oligosaccharide. Thus, the presentinvention has been completed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0025]FIG. 1: a figure which illustrates the ¹H-NMR spectrum of thesulfated fucan oligosaccharide 1-(1) according to the present invention.

[0026]FIG. 2: a figure which illustrates the ¹³C-NMR spectrum of thesulfated fucan oligosaccharide 1-(1) according to the present invention.

[0027]FIG. 3: a figure which illustrates the mass spectrum of thesulfated fucan oligosaccharide 1-(1) according to the present invention.

[0028]FIG. 4: a figure which illustrates the ¹H-NMR spectrum of thesulfated fucan oligosaccharide 1-(2) according to the present invention.

[0029]FIG. 5: a figure which illustrates the ¹³C-NMR spectrum of thesulfated fucan oligosaccharide 1-(2) according to the present invention.

[0030]FIG. 6: a figure which illustrates the mass spectrum of thesulfated fucan oligosaccharide 1-(2) according to the present invention.

[0031]FIG. 7: a figure which illustrates the ¹H-NMR spectrum of thesulfated fucan oligosaccharide 1-(3) according to the present invention.

[0032]FIG. 8: a figure which illustrates the ¹³C-NMR spectrum of thesulfated fucan oligosaccharide 1-(3) according to the present invention.

[0033]FIG. 9: a figure which illustrates the mass spectrum of thesulfated fucan oligosaccharide 1-(3) according to the present invention.

[0034]FIG. 10: a figure which illustrates the ¹H-NMR spectrum of thesulfated fucan oligosaccharide 1-(4) according to the present invention.

[0035]FIG. 11: a figure which illustrates the ¹³C-NMR spectrum of thesulfated fucan oligosaccharide 1-(4) according to the present invention.

[0036]FIG. 12: a figure which illustrates the mass spectrum of thesulfated fucan oligosaccharide 1-(4) according to the present invention.

[0037]FIG. 13: a figure which illustrates the ¹H-NMR spectrum of thesulfated fucan oligosaccharide 2-(1)-2 according to the presentinvention.

[0038]FIG. 14: a figure which illustrates the ¹³C-NMR spectrum of thesulfated fucan oligosaccharide 2-(1)-2 according to the presentinvention.

[0039]FIG. 15: a figure which illustrates the mass spectrum of thesulfated fucan oligosaccharide 2-(1)-2 according to the presentinvention.

[0040]FIG. 16: a figure which illustrates the ¹H-NMR spectrum of thesulfated fucan oligosaccharide 2-(2) according to the present invention.

[0041]FIG. 17: a figure which illustrates the ¹³C-NMR spectrum of thesulfated fucan oligosaccharide 2-(2) according to the present invention.

[0042]FIG. 18: a figure which illustrates the mass spectrum of thesulfated fucan oligosaccharide 2-(2) according to the present invention.

[0043]FIG. 19: a figure which illustrates the ¹H-NMR spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(1) according tothe present invention.

[0044]FIG. 20: a figure which illustrates the ¹³C-NMR spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(1) according tothe present invention.

[0045]FIG. 21: a figure which illustrates the mass spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(1) according tothe present invention.

[0046]FIG. 22: a figure which illustrates the ¹H-NMR spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(2) according tothe present invention.

[0047]FIG. 23: a figure which illustrates the ¹³C-NMR spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(2) according tothe present invention.

[0048]FIG. 24: a figure which illustrates the mass spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(2) according tothe present invention.

[0049]FIG. 25: a figure which illustrates the ¹H-NMR spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(3) according tothe present invention.

[0050]FIG. 26: a figure which illustrates the ¹³C-NMR spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(3) according tothe present invention.

[0051]FIG. 27: a figure which illustrates the mass spectrum of theLessonia nigrescens sulfated fucan oligosaccharide 1-(3) according tothe present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention will be explained in detail.

[0053] Although it is not intended to limit the present invention, forexample, a sulfated fucan derived from an alga belonging to Laminarialescan be used according to the present invention. This polysaccharide hasa sulfate group and fucose as main constituents. The main chain of asulfated fucan derived from an alga belonging to Laminariales iscomposed of L-fucose which is more acid-labile than general saccharides.Therefore, it can be readily converted into smaller molecules by heatingor acid treatment.

[0054] A sulfated fucan having the above-mentioned characteristics canbe used according to the present invention. Although there is nospecific limitation concerning the origin, for example, algae belongingto Laminariales such as Kjellmaniella crassifolia, Laminaria japonicaand Lessonia nigrescens are preferably used as raw materials becausetheir sulfated fucan contents are high.

[0055] The sulfated fucan-digesting enzyme according to the presentinvention acts on a sulfated fucan, a sulfated fucan oligosaccharide andthe like and hydrolyzes an α-L-fucosyl bond between fucoses in anendo-type manner to generate an oligosaccharide having L-fucose at itsreducing end.

[0056] The sulfated fucan oligosaccharide of the present invention is anoligosaccharide that is obtainable by allowing the sulfatedfucan-digesting enzyme according to the present invention to act on asulfated fucan and has L-fucose as a saccharide at the reducing end.

[0057] A water-soluble fraction extract is first obtained from a brownalga in order to produce the sulfated fucan used according to thepresent invention. In this case, it is preferable to obtain thewater-soluble fraction extract at pH 4-9 at a temperature of 100° C. orbelow in order to prevent the conversion of the sulfated fucan intosmaller molecules. Furthermore, amino acids, small molecule pigments orthe like in the extract can be efficiently removed by ultrafiltration.Activated carbon treatment or the like is effective for the removal ofhydrophobic substances.

[0058] A sulfated polysaccharide fraction from a brown alga can beobtained as described above. This fraction can be used as a sulfatedfucan fraction, for example, as a substrate for the sulfatedfucan-digesting enzyme according to the present invention. A more highlypure sulfated fucan can be obtained by separating the fraction using ananion exchange column. Both the sulfated polysaccharide fraction and thesulfated fucan purified using an anion exchange column can be used asraw materials for production of the sulfated fucan oligosaccharide ofthe present invention.

[0059] The main backbone of the sulfated fucan of the present inventionis represented by general formula (I) below. The sulfated fucans of thepresent invention include those of the general formula wherein n is aninteger of 1 or more, for example 1 to 20,000, preferably 1 to 10,000.The sulfated fucans of the present invention include those having astructure in which general formula (I) is continuously repeated andthose having a structure in which general formula (I) is discontinuouslyincluded being intervened by other structures as long as they are withinthe definition as described above.

[0060] wherein R is H or SO₃H, and at least one of Rs is SO₃H.;

[0061] The bacterial strain producing the sulfated fucan-digestingenzyme to be used according to the present invention is classified as abacterium belonging to genus Alteromonas according to the basicclassification as described in Bergey's Manual of DeterminativeBacteriology, Vol. 9 (1994). Classification of bacteria belonging togenus Alteromonas is recently re-organized. Therefore, it is notappropriate to classify the bacterium based only on the bacteriologicalproperties. The nucleotide sequence of 16S rDNA of this bacterial strainwas determined. Comparison of homologies with sequences of knownbacteria revealed that a bacterium belonging to genus Thalassomonas hasthe most homologous sequence. However, since the genetic distance is0.05 (change/average nucleotide position) or more, the bacterium was notdetermined to belong to this genus. Then, the present inventorsconcluded that this bacterium does not belong to known genera butbelongs to a novel genus based on the 16S rDNA sequence homology, anddesignated it as Fucanobacter lyticus SN-1009. As used herein,Alteromonas sp. SN-1009 is the same as Fucanobacter lyticus SN-1009.

[0062] This bacterial strain is designated as

[0063] Alteromonas sp. SN-1009 and deposited on Feb. 13, 1996 (date oforiginal deposit) at International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology, AIST TsukubaCentral 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566,Japan. under accession number FERM BP-5747 (transmitted to internationaldepositary authority under Budapest Treaty on Nov. 15, 1996). The 16SrDNA sequence of this bacterial strain is shown in SEQ ID NO:1.

[0064] Thus, the sulfated fucan-digesting enzyme used according to thepresent invention can be produced by culturing a bacterium that isdetermined to belong to the same genus as Fucanobacter lyticus SN-1009based on the 16S rDNA sequence. According to the present invention,Alteromonas sp. SN-1009, a spontaneous or artificial mutant ofAlteromonas sp. SN-1009 and microorganisms belonging to genusAlteromonas and genus Fucanobacter capable of producing the sulfatedfucan-digesting enzyme used according to the present invention can beutilized.

[0065] The sulfated fucan-digesting enzyme to be used according to thepresent invention can be obtained from the above-mentioned microorganismaccording to the method as described in Example 1.

[0066] The sulfated fucan oligosaccharide of the present invention canbe prepared by allowing a sulfated fucan-digesting enzyme to act on asulfated fucan-containing material. For example, a partially purifiedpreparation of sulfated fucan, a sulfated fucose-containingpolysaccharide fraction derived from a brown alga, a product obtained byextracting a brown alga with an aqueous solvent, or a brown alga itselfcan be preferably used as a sulfated fucan-containing material.

[0067] For preparing the sulfated fucan oligosaccharide of the presentinvention, a sulfated fucan or a sulfated fucan-containing material maybe dissolved according to a conventional method. The sulfated fucan ofthe present invention or the sulfated fucan-containing material may bedissolved in the solution at the maximal concentration. However, theconcentration is usually selected taking its operationality and theamount of the sulfated fucan-digesting enzyme according to the presentinvention used in a reaction into consideration. The solvent for thesulfated fucan may be selected from water, buffers and the likedepending on the objects. Usually, the pH of the solution is nearlyneutral. The enzymatic reaction is usually carried out at about 30° C.The molecular weight of the sulfated fucan oligosaccharide can becontrolled by adjusting the ratio or amount of the sulfatedfucan-digesting enzyme according to the present invention used in thereaction, the composition of the reaction mixture, the reaction time andthe like. The sulfated fucan oligosaccharide of the present inventionhaving more homogeneous molecular weight distribution or morehomogeneous charge density distribution can be prepared by subjectingthe sulfated fucan oligosaccharide of the present invention obtained asdescribed above to molecular weight fractionation or fractionation usingan anion exchange column. A conventional means for molecular weightfractionation can be applied. For example, gel filtration or molecularweight fractionation membrane may be used. Optionally, the smallermolecules may be further purified using ion-exchange resin treatment,active carbon treatment or the like, or they may be desalted, sterilizedor lyophilized. Thus, the sulfated fucan oligosaccharide of the presentinvention having a structure so homogeneous that one can determine thestructure by NMR analysis as described below can be obtained.

[0068] Although it is not intended to limit the present invention, forexample, a sulfated fucan oligosaccharide having a chemical structureselected from the group consisting of general formulas (II), (III),(XIII), (XIV),

[0069] wherein R is H or SO₃H and at least one of Rs is SO₃H in allformulas above, or a salt thereof exemplifies the sulfated fucanoligosaccharide of the present invention.

[0070] The sulfated fucan oligosaccharides of the present invention havesulfate groups within the molecules, which groups react with variousbases to form salts. The sulfated fucan oligosaccharide of the presentinvention is stable when it is in a form of salt. It is usually providedin a form of sodium and/or potassium and/or calcium salt. The sulfatedfucan oligosaccharide of the present invention in a free form can bederived from a salt thereof by utilizing cation-exchange resin such asDowex 50W. Optionally, it can be subjected to conventional salt-exchangeto convert it into any one of various desirable salts.

[0071] Pharmaceutically acceptable salts can be used as the salts of thesulfated fucan oligosaccharide of the present invention. Examples of thesalts include salts with alkaline metals such as sodium and potassiumand alkaline earth metals such as calcium, magnesium and zinc as well asammonium salts.

[0072] Additionally, the sulfated fucan oligosaccharide of the presentinvention can be used as a reagent for glycotechnology. For example, asubstance which is very useful as a reagent for glycotechnology (e.g.,which can be used as a fluorescence-labeled standard for the sulfatedfucan oligosaccharide) can be provided by subjecting the oligosaccharideto 2-aminopyridine (PA)-labeling according to the method as described inJP-B 5-65108 to prepare a PA-labeled oligosaccharide.

EXAMPLES

[0073] The following examples further illustrate the present inventionin detail but are not to be construed to limit the scope thereof.

[0074] In the following examples, the molecular weights of theoligosaccharides of sulfated fucan are their average molecular weightsthat were calculated from the results of mass analyses of them.

Referential Example 1 Preparation of Fucoidan from Kjellmaniellacrassifolia

[0075] 2 kg of dried cultured Kjellmaniella crassifolia was disruptedusing a cutter mill (Masuko Sangyo) equipped with a screen having a porediameter of 1 mm and suspended in 20 L of 80% ethanol. The suspensionwas stirred at 25° C. for 3 hours and filtered through a filter paper.The residue was suspended in 40 L of 30 mM phosphate buffer (pH 6.5)containing 100 mM sodium chloride. The suspension was treated at 95° C.for 2 hours and filtered through a stainless steel screen having a porediameter of 106 μm. 200 g of active carbon, 4.5 L of ethanol and 12,000U of alginate lyase K (Nagase Biochemicals) were added to the filtrate.The mixture was stirred at 25° C. for 20 hours, and then centrifuged.The supernatant was concentrated to 4 L using an ultrafiltration deviceequipped with hollow fibers with exclusion molecular weight of 100,000,centrifuged to remove insoluble substances and allowed to stand at 5° C.for 24 hours. The formed precipitate was removed by centrifugation. Thesupernatant was subjected to solvent exchange for 100 mM sodium chlorideusing an ultrafiltration device. The solution was cooled to 4° C. orbelow, and the pH was adjusted to 2.0 with hydrochloric acid. The formedprecipitate was removed by centrifugation. The pH of the supernatant wasadjusted to 8.0 with sodium hydroxide. The supernatant was concentratedto 4 L. The concentrate was subjected to solvent exchange for 20 mMsodium chloride using an ultrafiltration device. Insoluble substances inthe solution were removed by centrifugation. The supernatant was thenlyophilized to obtain 76 g of a dried fucoidan preparation fromKjellmaniella crassifolia.

Referential Example 2 Preparation of Sulfated Fucan Fraction

[0076] 7 g of the dried fucoidan preparation as described in ReferentialExample 1 was dissolved in 700 ml of 20 mM imidazole-hydrochloridebuffer (pH 8.0) containing 50 mM sodium chloride and 10% ethanol andcentrifuged to remove insoluble substances. The supernatant was loadedonto a DEAE-Cellulofine A-800 column (11.4×48 cm) equilibrated with thesame buffer. After washing with the same buffer, elution was thencarried out with a gradient of 50 mM to 1.95 M sodium chloride. Eachfraction contained 250 ml of the eluate. The total sugar content and theuronic acid content of each fraction were measured according to thephenol-sulfuric acid method and the carbazole-sulfuric acid method,respectively. The fractions 43-49, 50-55 and 56-67 were combined,desalted by electrodialysis and lyophilized. 340 mg, 870 mg and 2.64 gof dried products were obtained from the fractions 43-49, 50-55 and56-67, respectively. The fraction obtained from the fractions 56-67 wasused as a sulfated fucan fraction.

Referential Example 3 Method for Measuring Activity of SulfatedFucan-Digesting Enzyme

[0077] 12 μl of a 2.5% solution of the sulfated fucan fraction, 60 μl of50 mM imidazole-hydrochloride buffer (pH 7.5), 9 μl of 4 M sodiumchloride, 6 μl of 1 M calcium chloride, 21 μl of water and 12 μl of asolution of the sulfated fucan-digesting enzyme according to the presentinvention were mixed together. After reacting at 30° C. for 3 hours, thereaction mixture was treated at 100° C. for 10 minutes. Aftercentrifugation, 100 μl of the supernatant was analyzed using HPLC todetermine the degree of conversion into smaller molecules. As controls,a reaction mixture obtained by a reaction in which the buffer used fordissolving the sulfated fucan-digesting enzyme solution was used inplace of the digesting enzyme solution and a reaction mixture obtainedby a reaction in which water was used in place of the sulfated fucanfraction were similarly analyzed using HPLC.

[0078] One unit of an activity of sulfated fucan-digesting enzyme isdefined as an amount of an enzyme that cleaves fucosyl bonds in 1 μmolof a sulfated fucan in 1 minute in the above-mentioned reaction system.The activity of sulfated fucan-digesting enzyme was calculated accordingto the following equation:

{(12×1000×2.5/100)MG}×{(MG/M)−1}×{1/(180×0.012)}=U/ml

[0079] 12×1000×2.5/100: sulfated fucan fraction added (μg);

[0080] MG: the average molecular weight of the sulfated fucan as asubstrate;

[0081] M: the average molecular weight of the reaction product;

[0082] (MG/M)-1: the number of sites cleaved by the enzyme in onesulfated fucan molecule;

[0083] 180: the reaction time (minutes); and

[0084] 0.012: the volume of the enzyme solution (ml).

[0085] The HPLC was carried out as follows:

[0086] Instrument: L-6200 (Hitachi);

[0087] Column: OHpak SB-806HQ (8×300 mm; Showa Denko);

[0088] Eluent: 50 mM sodium chloride containing 5 mM sodium azide;

[0089] Detection: differential refractive index detector (Shodex R1-71,Showa Denko);

[0090] Flow rate: 1 ml/minute; and

[0091] Column temperature: 25° C.

[0092] The following procedure was carried out in order to determine theaverage molecular weight of the reaction product. Commercially availablepullulan (STANDARD P-82, Showa Denko) of which the molecular weight wasknown was analyzed under the same conditions as those for theabove-mentioned HPLC analysis. The relationship between the molecularweight of pullulan and retention time was expressed as a curve, whichwas used as a standard curve for determining the molecular weight of thereaction product. The amount of protein was determined by measuring theabsorbance of the enzyme solution at 280 nm. The calculation was carriedout assuming the absorbance of a solution containing a protein at aconcentration of 1 mg/ml as 1.0.

Example 1 Preparation of Sulfated Fucan-Digesting Enzyme

[0093]Fucanobacter lyticus SN-1009 was inoculated into 4 ml of a mediumconsisting of artificial seawater (Jamarine Laboratory) (pH 8.2)containing 0.2% fucoidan prepared as described in Referential Example 1and 0.3% peptone which had been autoclaved at 120° C. for 20 minutes,and cultured at 25° C. for 24 hours to prepare a seed culture. The seedculture was inoculated into 600 ml of a medium consisting of artificialseawater (pH 8.2) containing 0.25% glucose, 1% peptone, 0.05% yeastextract and antifoaming agent (KM70, Shin-Etsu Chemical) in a 2-LErlenmeyer flask which had been autoclaved at 120° C. for 20 minutes,and cultured at 25° C. for 20 hours. 20 L of a medium consisting ofartificial seawater (pH 8.2) containing 1% peptone, 0.02% yeast extractand antifoaming agent (KM70, Shin-Etsu Chemical) in a 30-L jar fermentorwhich had been autoclaved at 120° C. for 20 minutes was mixed with thefucoidan prepared as described in Referential Example 1 which had beentreated at 100° C. for 20 minutes. The culture was inoculated into themixture and cultured at 25° C. for 28 hours. After cultivation, theculture was centrifuged to collect cells and a culture supernatant.

[0094] The culture supernatant was concentrated using an ultrafiltrationdevice equipped with hollow fibers with exclusion molecular weight of10,000. The concentrate was subjected to solvent exchange for 20 mMTris-hydrochloride buffer (pH 8.2) containing 10 mM calcium chloride and150 mM sodium chloride and centrifuged to obtain a supernatant.

[0095] The supernatant was loaded onto a 2-L DEAE-Cellulofine A-800column equilibrated with the same buffer. After washing with the samebuffer, elution was then carried out with a gradient of 150 mM to 400 mMsodium chloride such that each fraction contained 63 ml of the eluate tocollect an active fraction.

[0096] The active fraction was concentrated using an ultrafiltrationdevice equipped with hollow fibers with exclusion molecular weight of10,000. The concentrate was subjected to solvent exchange for 20 mMTris-hydrochloride buffer (pH 8.2) containing 10 mM calcium chloride and100 mM sodium chloride. The enzyme solution was loaded onto a 200-mlDEAE-Cellulofine A-800 column equilibrated with the same buffer. Afterwashing with the same buffer, elution was then carried out with agradient of 100 mM to 300 mM sodium chloride such that each fractioncontained 19 ml of the eluate to collect an active fraction.

[0097] The active fraction was concentrated using an ultrafiltrationdevice equipped with an ultrafiltration membrane with exclusionmolecular weight of 10,000. Sodium chloride at a final concentration of4 M was added thereto. The solution was loaded onto a Phenyl-SepharoseCL-4B column equilibrated with 20 mM Tris-hydrochloride buffer (pH 8.0)containing 100 mM calcium chloride and 4 M sodium chloride. Afterwashing with the same buffer, elution was then carried out with agradient of 4 M to 1 M sodium chloride such that each fraction contained9.4 ml of the eluate. Thus, a purified preparation of sulfatedfucan-digesting enzyme was obtained.

Example 2 Preparation of Sulfated Fucan Oligosaccharide Using SulfatedFucan-Digesting Enzyme, and Purification and Structural Analysis Thereof

[0098] (1) Preparation

[0099] 200 g of dried cultured Kjellmaniella crassifolia was soaked in10 L of 18 mM imidazole-hydrochloride buffer (pH 7.0) containing 45 mMcalcium chloride, 500 mM sodium chloride and 9% ethanol. 30 U of thesulfated fucan-digesting enzyme was added thereto. The mixture wasstirred at room temperature for 2 days and filtered through a filterpaper. A small molecule fraction recovered from the filtrate using anultrafiltration device equipped with hollow fibers with exclusionmolecular weight of 100,000 was designated as a sulfated fucanoligosaccharide fraction 1.

[0100] (2) Purification

[0101] The sulfated fucan oligosaccharide fraction 1 obtained in Example2-(1) was desalted using a desalting apparatus (Micro Acilyzer G3, AsahiKasei) and concentrated using a rotary evaporator. Imidazole and sodiumchloride were added to the sulfated fucan oligosaccharide solution 1 atfinal concentrations of 10 mM and 300 mM, respectively. The resultingmixture was loaded onto a 1-L DEAE-Cellulofine A-800 column equilibratedwith 10 mM imidazole-hydrochloride buffer (pH 6.0) containing 300 mMsodium chloride. After adequately washing with the same buffer, elutionwas then carried out with a gradient of 300 mM to 1200 mM sodiumchloride. The total sugar content and the total uronic acid content ofeach of the eluted fractions were measured according to thephenol-sulfuric acid method and the carbazole-sulfuric acid method,respectively. As a result, the eluted fractions formed at least fourdistinct peaks. The fractions in the respective peaks were subjected tomass spectrometric analyses. Determination of the saccharidecompositions for the respective peaks showed that they contained onlyfucose but did not contain uronic acid. Composition of theoligosaccharides having the respective masses estimated based on thesaccharide compositions are shown in Table 1. TABLE 1 Composition ofoligosaccharide Molecular Sulfate Peak no. weight Fucose group Sodium1-(1) 1914 6 10 10 1812 6 9 9 1564 5 8 8 1-(2) 2264 7 12 12 2162 7 11 111914 6 10 10 2016 6 11 11 1-(3) 2366 7 13 13 2264 7 12 12 2016 6 11 111914 6 10 10 2162 7 11 11 1-(4) 3460 11 18 18 3358 11 17 17 3110 10 1616

[0102] The fractions constituting each peak were combined, concentratedusing an evaporator and purified as follows:

[0103] Column: YMC Pack Polyamine II (20×250 mm, YMC);

[0104] Flow rate: 8 ml/minute;

[0105] Column temperature: 30° C.;

[0106] Equilibration solution: 0.5 M sodium dihydrogenphosphatecontaining 10% acetonitrile;

[0107] Elution solution: gradient from 0.5 M sodium dihydrogenphosphatecontaining 10% acetonitrile to 1.5 M sodium dihydrogenphosphatecontaining 10% acetonitrile; as for peak number 1-(4), the gradient forthe elution was from 787.5 mM sodium dihydrogenphosphate containing 10%acetonitrile to 1462.5 mM sodium dihydrogenphosphate containing 10%acetonitrile;

[0108] Fractionation: 4 ml/fraction; and

[0109] Detection: the phenol-sulfuric acid method.

[0110] Fractions obtained by the column chromatography were subjected tomass spectrometric analyses. As a result, substances having masses of1914, 2264, 2366 and 3460 were observed as main peaks for the peak nos.1-(1), 1-(2), 1-(3) and 1-(4), respectively. Fractions constituting eachpeak were combined, loaded onto a Cellulofine GCL-25 column equilibratedwith 10% ethanol and eluted using 10% ethanol for desalting. Thus, thesulfated fucan oligosaccharides 1-(1) to 1-(4) of the present inventionwere obtained.

[0111] (3) Structural Analysis

[0112] The sulfated fucan oligosaccharides 1-(1) to 1-(4) of the presentinvention obtained in Example 2-(2) were subjected to analyses ofsaccharides at the reducing ends and saccharide compositions accordingto a fluorescence labeling method using 2-aminopyridine. As a result,the saccharide at the reducing end of and the sole saccharideconstituting each of the oligosaccharides 1-(1) to 1-(4) were determinedto be fucose. Next, determination of the sulfuric acid content (measuredaccording to the turbidinetric method using barium chloride) and theuronic acid content (measured according to the carbazole-sulfuric acidmethod), and NMR analysis using a nuclear magnetic resonance apparatusJNM α-500 (Nippon Denshi) were carried out. Samples to be analyzed weresubjected to structural analyses after exchange for heavy wateraccording to a conventional method. Bonds of constituting saccharideswere analyzed using the HMBC method, a method for ¹H-detection ofheteronuclei. The DQF-COSY method and the HOHAHA method were used foridentification in ¹H-NMR. The HSQC method was used for identification in¹³C-NMR.

[0113] Physical properties of the oligosaccharides 1-(1) to 1-(4) areshown below.

[0114] (a) Physical Properties of the Oligosaccharide 1-(1)

[0115] The results for mass spectrometric analysis and identification inNMR analysis are shown below. The ¹H-NMR spectrum, ¹³C-NMR spectrum andmass spectrum of the sulfated fucan oligosaccharide 1-(1) of the presentinvention are illustrated in FIGS. 1, 2 and 3, respectively. In FIGS. 1and 2, the vertical axes represent the signal intensity and thehorizontal axes represent the chemical shift value (ppm). In FIG. 3, thevertical axis represents the relative intensity and the horizontal axisrepresents the m/z value.

[0116] Molecular weight: 1914

[0117] MS m/z 455.0 [M-4Na⁺]⁴⁻, 614.8 [M-3Na⁺]³⁻, 933.8 [M-2Na⁺]²⁻

[0118] Results of ¹H-NMR and ¹³C-NMR analyses are shown in Tables 2 and3. TABLE 2 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 91.3 5.41, d, 3.4 F1-2 75.94.39, dd, 3.4, 10.1 F1-3 75.9 4.14, dd, 2.4, 10.1 F1-4 81.1 4.81, d, 2.4F1-5 67.6 4.20, q, 6.4 F1-6 16.8 1.16, d, 6.4 F2-1 99.7 5.25, d, 1.8F2-2 75.2 4.43, m F2-3 72.5 4.43, m F2-4 78.8 4.75, br-s F2-5 68.3 4.27,q, 6.7 F2-6 17.0 1.18, d, 6.7 F3-1 91.0 5.22, br-s F3-2 75.8 4.10, d,5.2 F3-3 74.7 4.27, dd, 5.2, 2.5 F3-4 74.0 4.73, dd, 2.5, 5.5 F3-5 71.94.32, m, 5.5, 6.1 F3-6 14.2 1.45, d, 6.1

[0119] TABLE 3 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F4-1 98.5 5.32, d, 3.1 F4-2 74.04.45, m F4-3 71.0 4.27, m F4-4 78.9 4.76, br-s F4-5 68.3 4.29, q, 6.7F4-6 17.0 1.18, d, 6.7 F5-1 96.1 5.29, d, 3.4 F5-2 75.9 4.35, dd, 3.4,10.7 F5-3 67.4 4.27, dd, 2.4, 10.7 F5-4 82.3 4.60, d, 2.4 F5-5 67.44.38, q, 6.4 F5-6 17.0 1.16, d, 6.4 F6-1 100.5 5.18, d, 4.0 F6-2 66.73.84, dd, 4.0, 10.4 F6-3 78.8 4.45, dd, 3.1, 10.4 F6-4 71.1 4.07, d, 3.1F6-5 67.9 4.06, q, 6.4 F6-6 16.5 1.13, d, 6.4

[0120] Saccharide composition: L-fucose (6 molecules)

[0121] Sulfate group: 10 molecules

[0122] Sodium: 10 molecules

[0123] The numbers for peak identification in ¹H-NMR and ¹³C-NMR are asindicated in formula (IV) below:

[0124] (b) Physical Properties of the Oligosaccharide 1-(2)

[0125] The results for mass spectrometric analysis and identification inNMR analysis are shown below. The ¹H-NMR spectrum, ¹³C-NMR spectrum andmass spectrum of the sulfated fucan oligosaccharide 1-(2) of the presentinvention are illustrated in FIGS. 4, 5 and 6, respectively. In FIGS. 4and 5, the vertical axes represent the signal intensity and thehorizontal axes represent the chemical shift value (ppm). In FIG. 6, thevertical axis represents the relative intensity and the horizontal axisrepresents the m/z value.

[0126] Molecular weight: 2264

[0127] MS m/z 354.2 [M-6Na⁺]⁶⁻, 429.8 [M-5Na⁺]⁵⁻, 543.0 [M-4Na⁺]⁴⁻,731.6 [M-3Na⁺]³⁻

[0128] Results of ¹H-NMR and ¹³C-NMR analyses are shown in Tables 4 and5. TABLE 4 Chemical shift value (ppm) ¹³C-NMR ¹H-NMR F1-1 90.3 5.41, d,3.4 F1-2 75.7 4.38, dd, 3.4, 9.8 F1-3 74.9 4.22, m F1-4 80.2 4.80, br-sF1-5 67.5 4.23, q, 6.7 F1-6 16.0 1.19, d, 6.7 F2-1 99.3 5.31, d, 3.4F2-2 75.2 4.40, m F2-3 76.9 4.29, m F2-4 81.5 4.83, d, 2.8 F2-5 68.54.22, q, 6.4 F2-6 16.4 1.16, d, 6.4 F3-1 99.7 5.29, br-s F3-2 74.0 4.41,m F3-3 71.1 4.41, m F3-4 78.2 4.75, m F3-5 67.4 4.34, q, 6.7 F3-6 16.51.22, d, 6.7 F4-1 89.6 5.26, br-s F4-2 74.4 4.07, m F4-3 74.4 4.27, mF4-4 73.3 4.73, dd, 2.8, 5.8 F4-5 71.2 4.36, m, 5.8, 6.4 F4-6 13.5 1.44,d, 6.4

[0129] TABLE 5 Chemical shift value (ppm) ¹³C-NMR ¹H-NMR F5-1 98.3 5.31,d, 3.4 F5-2 73.4 4.45, dd, 3.4, 10.1 F5-3 70.4 4.26, m F5-4 78.2 4.76,br-s F5-5 67.6 4.33, q, 6.4 F5-6 16.4 1.23, d, 6.4 F6-1 95.2 5.29, d,3.7 F6-2 75.2 4.36, m F6-3 66.8 4.27, m F6-4 81.7 4.60, d, 2.8 F6-5 66.84.38, q, 6.7 F6-6 16.4 1.16, d, 6.7 F7-1 100.1 5.17, d, 4.3 F7-2 66.13.86, dd, 4.3, 10.4 F7-3 78.3 4.45, dd, 3.7, 10.4 F7-4 70.5 4.07, d, 3.7F7-5 67.4 4.05, q, 6.4 F7-6 16.0 1.13, d, 6.4

[0130] Saccharide composition: L-fucose (7 molecules)

[0131] Sulfate group: 12 molecules

[0132] Sodium: 12 molecules

[0133] The numbers for peak identification in ¹H-NMR and ³C-NMR are asindicated in formula (V) below:

[0134] An activity of inducing HGF production as determined according tothe method described in Example 1-(2) in WO 00/62785 was observed forthe sulfated saccharide of formula (V).

[0135] (c) Physical Properties of the Oligosaccharide 1-(3)

[0136] The results for mass spectrometric analysis are shown below. The¹H-NMR spectrum, ¹³C-NMR spectrum and mass spectrum of the sulfatedfucan oligosaccharide 1-(3) of the present invention are illustrated inFIGS. 7, 8 and 9. In FIGS. 7 and 8, the vertical axes represent thesignal intensity and the horizontal axes represent the chemical shiftvalue (ppm) In FIG. 9, the vertical axis represents the relativeintensity and the horizontal axis represents the m/z value.

[0137] Molecular weight: 2366

[0138] MS m/z 371.4 [M-6Na⁺]⁶⁻, 450.3 [M-5Na⁺]⁵⁻, 568.6 [M-4Na⁺]⁴⁻,765.7 [M-3Na⁺]³⁻

[0139] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Tables 6and 7. TABLE 6 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 89.6 5.42, d, 2.5 F1-2 75.34.40, m F1-3 73.9 4.28, m F1-4 78.6 4.78, m F1-5 67.5 4.24, m F1-6 15.91.24, d, 6.5 F2-1 98.3 5.34, d, 3.5 F2-2 74.8 4.43, m F2-3 76.0 4.29, mF2-4 80.8 4.83, m F2-5 68.1 4.20, m F2-6 15.9 1.18, d, 7.0 F3-1 99.15.29, m F3-2 73.8 4.45, m F3-3 70.8 4.45, m F3-4 77.5 4.73, m F3-5 66.84.31, m F3-6 15.9 1.25, d, 6.5

[0140] TABLE 7 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F4-1 89.2 5.27, br-s F4-2 74.14.09, d, 9.5 F4-3 74.3 4.26, m F4-4 72.8 4.74, m F4-5 70.8 4.36, m F4-613.3 1.46, d, 7.0 F5-1 97.3 5.31, d, 3.5 F5-2 73.1 4.47, dd, 10.0, 3.5F5-3 69.8 4.27, m F5-4 77.5 4.75, m F5-5 67.0 4.34, m F5-6 15.9 1.20, d,6.0 F6-1 94.6 5.29, m F6-2 74.9 4.38, m F6-3 66.6 4.29, m F6-4 81.34.60, m F6-5 66.4 4.38, m F6-6 15.4 1.17, d, 6.5 F7-1 99.3 5.22, d, 4.0F7-2 65.9 3.91, dd, 11.0, 4.0 F7-3 75.3 4.52, dd, 11.0, 3.0 F7-4 79.04.80, d, 3.0 F7-5 66.6 4.16, m F7-6 15.9 1.20, d, 6.0

[0141] Saccharide composition: L-fucose (7 molecules)

[0142] Sulfate group: 13 molecules

[0143] Sodium: 13 molecules

[0144] The numbers for signal assignment in ¹H-NMR and

[0145]¹³C-NMR are as indicated in formula (VI) below:

[0146] (d) Physical Properties of the Oligosaccharide 1-(4)

[0147] The results for mass spectrometric analysis are shown below. The¹H-NMR spectrum, ¹³C-NMR spectrum and mass spectrum of the sulfatedfucan oligosaccharide 1-(4) of the present invention are illustrated inFIGS. 10, 11 and 12. In FIGS. 10 and 11, the vertical axes represent thesignal intensity and the horizontal axes represent the chemical shiftvalue (ppm). In FIG. 12, the vertical axis represents the relativeintensity and the horizontal axis represents the m/z value.

[0148] Molecular weight: 3460

[0149] MS m/z 409.6 [M-8Na⁺]⁸⁻, 471.4 [M-7Na⁺]⁷⁻, 553.8 [M-6Na⁺]⁶⁻,669.3 [M-5Na⁺]⁵⁻, 842.3 [M-4Na⁺]⁴⁻, 1130.8 [M-3Na⁺]³⁻

[0150] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Tables 8to 10. TABLE 8 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 90.3 5.39, d, 3.3 F1-2 75.54.37, m F1-3 76.0 4.21, m F1-4 79.8 4.77, m F1-5 67.5 4.19, m F1-6 16.51.15, m F2-1 99.8 5.28, m F2-2 73.6 4.41, m F2-3 73.6 4.41, m F2-4 78.14.74, m F2-5 68.7 4.25, m F2-6 16.8 1.20, m F3-1 98.7 5.31, m F3-2 74.84.46, m F3-3 73.6 4.35, m F3-4 78.1 4.74, m F3-5 67.7 4.25, m F3-6 16.81.20, m F4-1 90.2 5.29, m F4-2 74.8 4.04, m F4-3 74.9 4.27, m F4-4 74.34.74, m F4-5 68.1 4.27, q, 6.5 F4-6 14.0 1.39, d, 6.5

[0151] TABLE 9 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F5-1 99.2 5.34, d, 3.5 F5-2 74.34.42, m F5-3 74.3 4.21, m F5-4 81.5 4.81, m F5-5 68.7 4.20, m F5-6 16.61.18, m F6-1 98.7 5.31, m F6-2 75.3 4.46, m F6-3 71.1 4.35, m F6-4 78.14.74, m F6-5 67.7 4.25, m F6-6 16.7 1.19, m F7-1 90.2 5.29, m F7-2 74.84.04, m F7-3 74.9 4.27, m F7-4 74.3 4.74, m F7-5 67.9 4.27, q, 7.0 F7-614.0 1.43, d, 7.0

[0152] TABLE 10 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F8-1 96.0 5.30, m F8-2 75.34.46, m F8-3 71.1 4.35, m F8-4 78.1 4.81, m F8-5 67.1 4.43, m F8-6 16.71.19, m F9-1 96.0 5.29, m F9-2 75.5 4.33, m F9-3 67.1 4.27, m F9-4 82.04.58, d, 2.8 F9-5 67.1 4.34, q, 6.5 F9-6 16.5 1.15, d, 6.5 F10-1 99.85.19, d, 4.2 F10-2 66.3 3.85, dd, 4.2, 10.4 F10-3 78.5 4.43, m F10-470.8 4.05, br-s F10-5 67.5 4.03, q, 6.6 F10-6 16.1 1.12, d, 6.6 F11-199.8 5.21, d, 4.2 F11-2 66.3 3.85, dd, 4.2, 10.4 F11-3 78.5 4.44, mF11-4 70.8 4.06, br-s F11-5 67.5 4.08, q, 6.6 F11-6 16.1 1.12, d, 6.6

[0153] Saccharide composition: L-fucose (11 molecules)

[0154] Sulfate group: 18 molecules

[0155] Sodium: 18 molecules

[0156] The numbers for signal assignment in ¹H-NMR and

[0157]¹³C-NMR are as indicated in formula (VII) below:

Example 3 Preparation of Sulfated Fucan Oligosaccharide Using SulfatedFucan-Digesting Enzyme, and Purification and Structural Analysis Thereof

[0158] (1) Preparation

[0159] Chips were prepared from dried cultured Kjellmaniella crassifoliausing a cutter mill (Masuko Sangyo) equipped with a screen having a porediameter of 1 mm. 250 g of the Kjellmaniella crassifolia chips weresuspended in 5 L of 80% ethanol. The suspension was stirred at roomtemperature for 2 hours and filtered. The washing in 80% ethanol wasrepeated four times to obtain washed Kjellmaniella crassifolia chips.250 g of the washed chips were suspended in 5 L of 17 mMimidazole-hydrochloride buffer (pH 7.5) containing 125 mM calciumchloride, 250 mM sodium chloride and 10% ethanol. The suspension wasstirred at room temperature for 24 hours, filtered and centrifuged toobtain a Kjellmaniella crassifolia fucoidan solution. 10 U of thesulfated fucan-digesting enzyme was added to 1 L of the extract. Theresulting mixture was stirred at room temperature for three days, andthen filtered through a filter paper. A supernatant was obtained bycentrifuging the filtrate. A small molecule fraction recovered from thesupernatant using an ultrafiltration device equipped with hollow fiberswith exclusion molecular weight of 10,000 was designated as a sulfatedfucan oligosaccharide fraction 2.

[0160] (2) Purification

[0161] The sulfated fucan oligosaccharide fraction 2 obtained in Example3-(1) was loaded onto a 1-L DEAE-Cellulofine A-800 column equilibratedwith 10 mM imidazole-hydrochloride buffer (pH 6.5) containing 100 mMsodium chloride. After adequately washing with the same buffer, elutionwas then carried out with a gradient of 100 mM to 1 M sodium chloride.The total sugar content and the total uronic acid content of each of theeluted fractions were measured according to the phenol-sulfuric acidmethod and the carbazole-sulfuric acid method, respectively. As aresult, the eluted fractions formed two peaks. The fractions in therespective peaks were subjected to mass spectrometric analyses.Determination of the saccharide compositions for the respective peaksshowed that they contained only fucose but did not contain uronic acid.Compositions of the oligosaccharides having the respective massesestimated based on the saccharide compositions are shown in Table 11.TABLE 11 Composition of oligosaccharide Molecular Sulfate Peak no.weight Fucose group Sodium 2-(1) 1914 6 10 10 2264 7 12 12 2016 6 11 112-(2) 3110 10 16 16 2760 9 14 14 3360 11 17 17 4308 14 22 22 3958 13 2020

[0162] The fractions constituting each peak were pooled, concentratedusing an evaporator, and purified by the same method described inExample 2-(2).

[0163] Fractions obtained by YMC Pack Polyamine II column chromatographywere subjected to mass spectrometric analyses, and substances havingmasses of 1914 and 2016 from peak number 2-(1), and a substance having amass of 3110 from peak number 2-(2) were obtained as their main peak.

[0164] The fractions constituting each peak that contained having massesof 1914, 2016, and 3110 were polled, applied to a Cellulofine GCL-25column equilibrated with 10% ethanol and eluted using 10% ethanol todesalt. Thus the sulfated fucan oligosaccharides 2-(1)-1, 2-(1)-2, and2-(2) of the present invention were obtained.

[0165] (3) Structural Analyses

[0166] The sulfated fucan oligosaccharides 2-(1)-1, 2-(1)-2, and 2-(2)of the present invention obtained in Example 3-(2) were subjected toanalyses of the reducing terminal sugar and component sugar according toa fluorescence labeling method using 2-aminopyridine. As a result, thereducing terminal and the component sugar of them were only fucose.Next, their sulfate content (by the turbidimetric method using bariumchloride) and their uronic acid content (by the carbazole-sulfuric acidmethod) were determined. NMR analyses of them using a nuclear magneticresonance apparatus JNM α-500 (Nippon Denshi) were also carried out. Thelinkages among the constituting saccharides were analyzed using the HMBCmethod, a method for ¹H-detection of heteronuclei. The DQF-COSY methodand the HOHAHA method were used for the assignment of ¹H-NMR data, andthe HSQC method was used for the assignment of ¹³C-NMR data.

[0167] Physical properties of the sulfated fucan oligosaccharides2-(1)-1,2-(1)-2, and 2-(2) of the present invention are shown below.

[0168] (a) Physical Properties of the Sulfated Fucan Oligosaccharide2-(1)-1

[0169] The result of all the above analyses showed that the sulfatedfucan oligosaccharide 2-(1)-1 is the same substance as the sulfatedfucan oligosaccharide 1-(1) of the present invention.

[0170] (b) Physical Properties of the Sulfated Fucan Oligosaccharide2-(1)-2

[0171] The results for the mass spectrometric analysis and assignment inNMR analyses are shown below. The ¹H-NMR spectrum, ¹³C-NMR spectrum, andthe mass spectrum of this oligosaccharide are shown in FIGS. 13, 14, and15, respectively. In FIGS. 13 and 14, the vertical axes represent thesignal intensity and the horizontal axes represent the chemical shiftvalue (ppm). In FIG. 15, the vertical axis represents the relativeintensity of the signals and the horizontal axis represents the m/zvalue.

[0172] Molecular weight: 2016

[0173] MS m/z 313.1 [M-6Na⁺]⁶⁻, 380.3 [M-5Na⁺]⁵⁻, 481.1 [M-4Na⁺]⁴⁻,649.1 [M-3Na⁺]³⁻, 985.0 [M-2Na⁺]²⁻

[0174] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Tables 12and 13. TABLE 12 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 90.7 5.43, d, 3.6 F1-2 75.34.41, dd, 3.6, 10.1 F1-3 75.7 4.15, dd, 10.1, 2.4 F1-4 80.6 4.83, d, 2.4F1-5 67.0 4.22, q, 6.6 F1-6 16.5 1.18, d, 6.6 F2-1 99.2 5.27, d, 3.2F2-2 74.5 4.45, m F2-3 71.9 4.46, m F2-4 78.1 4.77, br-s F2-5 67.7 4.30,q, 6.6 F2-6 16.5 1.20, d, 6.6 F3-1 89.8 5.24, br-s F3-2 75.2 4.12, mF3-3 74.2 4.28, m F3-4 73.3 4.76, br-s F3-5 71.4 4.36, q, 6.0 F3-6 13.61.48, d, 6.0

[0175] TABLE 13 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F4-1 98.2 5.33, d, 3.3 F4-2 73.34.47, m F4-3 71.0 4.28, m F4-4 78.1 4.78, br-s F4-5 67.7 4.32, q, 6.6F4-6 16.5 1.20, d, 6.6 F5-1 94.8 5.31, d, 3.4 F5-2 75.3 4.38, m F5-366.9 4.28, m F5-4 81.7 4.62, d, 2.4 F5-5 66.8 4.40, q, 6.6 F5-6 16.51.18, d, 6.6 F6-1 100.1 5.22, d, 3.6 F6-2 66.3 3.89, dd, 3.6, 10.4 F6-375.7 4.52, dd, 10.4, 3.0 F6-4 79.2 4.82, d, 3.0 F6-5 67.2 4.18, q, 6.6F6-6 16.3 1.20, d, 6.6

[0176] Component sugar: only L-fucose (6 molecules)

[0177] Sulfate residues: 11 molecules

[0178] Sodium: 11 molecules

[0179] The numbers for signal assignment in ¹H-NMR and ³C-NMR analysesare as indicated in formula (VIII) below:

[0180] Physical Properties of the Oligosaccharide 2-(2)

[0181] The results of the mass spectrometric analysis are shown below,and the ¹H-NMR spectrum, ¹³C-NMR spectrum, and the mass spectrum of thesulfated fucan oligosaccharide 2-(2) of the present invention are shownin FIGS. 16, 17, and 18, respectively. In FIGS. 16 and 17, the verticalaxes represent the signal intensity and the horizontal axes representthe chemical shift value (ppm). In FIG. 18, the vertical axis representsthe relative intensity of the signals and the horizontal axis representsthe m/z value.

[0182] Molecular weight: 3111

[0183] MS m/z 365.8[M-8Na⁺]⁸⁻, 421.4[M-7Na⁺]⁷⁻, 495.2[M-6Na⁺]⁶⁻,599.1[M-5Na⁺]⁵⁻, 755.0[M-4Na⁺]⁴⁻, 1013.7[M-3Na⁺]³⁻

[0184] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Tables 14to 16. TABLE 14 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 90.8 5.44, d, 3.4 F1-2 75.74.44, dd, 3.4, 9.8 F1-3 75.9 4.19, dd, 9.8, 2.9 F1-4 80.3 4.84, d, 2.9F1-5 67.7 4.24, m F1-6 16.6 1.22, m F2-1 99.4 5.30, d, 3.0 F2-2 74.94.46, m F2-3 72.5 4.44, m F2-4 78.4 4.78, br-s F2-5 68.2 4.29, q, 6.6F2-6 17.1 1.25, d, 6.6 F3-1 91.2 5.29, br-s F3-2 74.9 4.11, m F3-3 75.04.28, m F3-4 74.5 4.78, br-s F3-5 68.4 4.31, q, 6.8 F3-6 14.5 1.43, d,6.8 F4-1 98.7 5.40, d, 3.5 F4-2 74.9 4.48, m F4-3 74.9 4.29, m F4-4 80.34.48, br-s F4-5 68.4 4.24, m F4-6 16.6 1.22, m

[0185] TABLE 15 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F5-1 98.0 5.35, d, 3.6 F5-2 73.84.51, m F5-3 71.2 4.39, m F5-4 78.4 4.78, m F5-5 68.2 4.29, m F5-6 16.91.23, m F6-1 90.5 5.34, br-s F6-2 74.9 4.11, m F6-3 75.0 4.29, m F6-474.5 4.78, br-s F6-5 68.4 4.31, q, 7.0 F6-6 14.2 1.48, d, 7.0 F7-1 95.95.33, m F7-2 73.9 4.48, m F7-3 71.2 4.37, m F7-4 78.2 4.78, m F7-5 67.34.46, m F7-6 16.8 1.23, m

[0186] TABLE 16 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F8-1 96.2 5.33, d, 3.3 F8-2 75.74.38, m F8-3 67.3 4.31, m F8-4 82.2 4.62, d, 3.1 F8-5 67.3 4.38, m F8-616.4 1.20, m F9-1 99.7 5.25, d, 4.4 F9-2 66.5 3.88, dd, 4.4, 10.2 F9-378.7 4.48, m F9-4 70.9 4.10, m F9-5 67.5 4.08, q, 6.6 F9-6 16.2 1.17, d,6.6 F10-1 99.7 5.26, d, 4.4 F10-2 66.5 3.90, dd, 4.4, 10.2 F10-3 78.74.49, m F10-4 70.9 4.11, m F10-5 67.5 4.12, q, 6.6 F10-6 16.3 1.17, d,6.6

[0187] Saccharide composition: L-fucose (10 molecules)

[0188] Sulfate group: 16 molecules

[0189] Sodium: 16 molecules

[0190] The numbers for signal assignment in ¹H-NMR and ¹³C-NMR analysesare as indicated in formula (IX) below:

Example 4 The Preparation of Laminaria japonica Sulfated FucanOligosaccharides Using Sulfated Fucan-Digesting Enzyme, theirPurification, and their Structural Analyses

[0191] (1) Preparation

[0192] Dried cultured Laminaria japonica was disrupted into chips usinga cutter mill (Masuko Sangyo) equipped with a screen having a porediameter of 1 mm. Five hundred grams of Laminaria japonica chips weresuspended in 4.5 L of 80% ethanol, stirred at room temperature for 24hours, and filtered. The residue was washed with 80% ethanol for threetimes by the same method as described above, and the washed Laminariajaponica chips were obtained. All the washed chips were suspended in 10L of 50 mM imidazole-HCl buffer (pH 8.0) containing 50 mM calciumchloride, 200 mM sodium chloride, and 10% ethanol, to the suspension wasadded 2 U of the sulfated fucan-digesting enzyme, stirred at roomtemperature for 5 days, filtered through filter paper, thus obtainedfiltrate was centrifuged, and the supernatant was fractionated using anultrafiltration device equipped with hollow fibers with exclusionmolecular weight of 10,000, and the low molecular weight fraction wasrecovered and designated as a Laminaria japonica sulfated fucanoligosaccharide fraction 1.

[0193] (2) Purification

[0194] The conductivity of the solution of the Laminaria japonicasulfated fucan oligosaccharide fraction 1 obtained in Example 4-(1) wasadjusted by adding 10% ethanol to the same conductivity of theequilibration buffer described below, and it was applied to 500 ml ofDEAE-Cellulofine A-800 column equilibrated with 20 mM imidazole-HClbuffer (pH 6.5) containing 200 mM sodium chloride and 10% ethanol, thenthe column was washed with the same buffer, and eluted with a lineargradient of sodium chloride from 200 mM to 1.2 M. For the elution, 4.5 Lof the buffer was used, and fractions of 50 ml were collected. The totalsugar content and the total uronic acid content of each of the elutedfractions were measured by the phenol-sulfuric acid method andcarbazole-sulfuric acid method, respectively. As a result, the elutedfractions formed 3 peaks. Fractions around the second peak (44-48) werepooled, concentrated to 40 ml by evaporator, applied to CellulofineGCL-25 column (4.1×90 cm) equilibrated with 10% ethanol, and eluted with10% ethanol. The total sugar of each of the eluted fractions wasdetermined by phenol-sulfuric acid method. The fractions in which sugarwas detected were pooled, concentrated to 8.4 ml by evaporator, andpurified by the condition described below.

[0195] Column: YMC Pack Polyamine II (20×250 mm, YMC)

[0196] Flow rate: 8 ml/minute

[0197] Column temperature: 30° C.

[0198] Equilibration solution: 875 mM sodium dihydrogenphosphatecontaining 10% acetonitrile

[0199] Elution: Gradient from 875 mM sodium dihydrogenphosphatecontaining 10% acetonitrile to 1.4 M sodium dihydrogenphosphatecontaining 10% acetonitrile

[0200] Fractionation: 4 ml/fraction

[0201] Detection: by the phenol-sulfuric acid method

[0202] Fractions obtained by the column chromatography shown above (3times, fraction number around 50-59) were pooled, dialyzed against 10%ethanol using a dialysis tube of which the exclusion molecular weight is3,500, concentrated to about 40 ml by evaporator, applied to CellulofineGCL-25 column equilibrated with 10% ethanol, and eluted with 10%ethanol.

[0203] The total sugar of each of the eluted fractions was determined bythe phenol-sulfuric acid method. The fractions around the main peak werepooled and purified again by Cellulofine GCL-25 as described above. Thefractions around the main peak of these eluted fractions were pooled,concentrated by Speed Vac, and lyophilized. Thus Laminaria japonicasulfated fucan oligosaccharide 1 was obtained.

[0204] (3) Structural Analyses

[0205] By the structural analyses of the oligosaccharide as shown inExample 2, the structure of Laminaria japonica sulfated fucanoligosaccharide 1 was determined to be the same structure as thesulfated fucan oligosaccharide 1-(3) shown in Example 2.

Example 5 Preparation, Purification, and Structural Analyses of Lessonianigrescence Sulfated Fucan Oligosaccharide

[0206] (1) Preparation

[0207] By the method shown in Referential Example 1, Lessonia nigrescensfucoidan was prepared from dried chips of Lessonia nigrescens.

[0208] Namely, 10 g of Lessonia nigrescens fucoidan was dissolved in 2 Lof 50 mM imidazole-HCl buffer (pH 8.0) containing 50 mM calciumchloride, 300 mM sodium chloride, and 10% ethanol. To the solution wasadded 1 U of sulfated fucan-digesting enzyme, stirred at roomtemperature for 40 hours, and recovered its low molecular weightfraction using an ultrafiltration device equipped with hollow fiberswith exclusion molecular weight of 10,000, and thus obtained fractionwas designated as a Lessonia nigrescens sulfated fucan oligosaccharidefraction 1.

[0209] (2) Purification

[0210]Lessonia nigrescens sulfated fucan oligosaccharide fraction 1obtained in (1) was applied to 1000 ml of DEAE-Cellulofine A-800 columnequilibrated with 10 mM imidazole-HCl buffer (pH 6.0) containing 200 mMsodium chloride and 10% ethanol, then the column was washed with thesame buffer, and eluted with a linear gradient of sodium chloride from200 mM to 700 mM. For the elution, 5 L of the buffer was used, andfractions of 56 ml were collected. The total sugar content and the totaluronic acid content of each of the eluted fractions were measured by thephenol-sulfuric acid method and carbazole-sulfuric acid method,respectively. As a result, the eluted fractions formed 7 peaks.Fractions around the each peak were pooled separately, concentrated to40 ml by evaporator (they are designated as Lessonia nigrescens sulfatedfucan oligosaccharides 1-(1) to 1-(7)), applied to Cellulofine GCL-25column (4.1×90 cm) equilibrated with 10% ethanol, and eluted with 10%ethanol. The total sugar of each of the eluted fractions was determinedby phenol-sulfuric acid method. The fractions in which sugar wasdetected were pooled, concentrated by evaporator, and purified by thecondition described below.

[0211] Column: YMC Pack Polyamine II (20×250 mm, YMC)

[0212] Flow rate: 8 ml/minute

[0213] Column temperature: 30° C.

[0214] Equilibration solution: For Lessonia nigrescens sulfated fucanoligosaccharides 1-(1), (2), and (3), 90 mM sodium dihydrogenphosphatecontaining 10% acetonitrile. For Lessonia nigrescens sulfated fucanoligosaccharide 1-(4) and (5), 630 mM sodium dihydrogenphosphatecontaining 10% acetonitrile. For Lessonia nigrescens sulfated fucanoligosaccharide 1-(6), 720 mM sodium dihydrogenphosphate containing 10%acetonitrile. For Lessonia nigrescens sulfated fucan oligosaccharide1-(7), 900 mM sodium dihydrogenphosphate containing 10% acetonitrile.Elution: For Lessonia nigrescens sulfated fucan oligosaccharides 1-(1),(2), and (3), with the gradient from 90 mM sodium dihydrogenphosphatecontaining 10% acetonitrile to 900 mM sodium dihydrogenphosphatecontaining 10% acetonitrile. For Lessonia nigrescens sulfated fucanoligosaccharides 1-(4) and (5), with the gradient from 630 mM sodiumdihydrogenphosphate containing 10% acetonitrile to 1260 mM sodiumdihydrogenphosphate containing 10% acetonitrile. For Lessonia nigrescenssulfated fucan oligosaccharide 1-(6), with the gradient from 720 mMsodium dihydrogenphosphate containing 10% acetonitrile to 1440 mM sodiumdihydrogenphosphate containing 10% acetonitrile. For Lessonia nigrescenssulfated fucan oligosaccharide 1-(7), with the gradient from 900 mMsodium dihydrogenphosphate containing 10% acetonitrile to 1620 mM sodiumdihydrogenphosphate containing 10% acetonitrile.

[0215] Fractionation: 4 ml/fraction

[0216] Detection: by the phenol-sulfuric acid method

[0217]Lessonia nigrescens sulfated fucan oligosaccharides from 1-(1) to1-(7) were fractionated by the column chromatography shown above, andthe fractions in which sugar was detected were pooled, concentrated toabout 40 ml by evaporator, applied to Cellulofine GCL-25 columnequilibrated with 10% ethanol, and eluted with 10% ethanol.

[0218] The total sugar of each of the eluted fractions was determined bythe phenol-sulfuric acid method. The fractions around the main peak werepooled and purified again by Cellulofine GCL-25 as described above. Asfor Lessonia nigrescens sulfated fucan oligosaccharide 1-(7), two mainpeaks were observed, therefore, the two peaks were designated asLessonia nigrescens sulfated fucan oligosaccharides 1-(7)-1 and 1-(7)-2.

[0219] The fractions around the main peak of each of these elutedfractions were pooled, concentrated by Speed Vac, and lyophilized. ThusLessonia nigrescens sulfated fucan oligosaccharides from 1-(1) to1-(7)-2 were obtained.

[0220] (3) Structural Analyses

[0221] The structural analyses of these oligosaccharides were carriedout as described in Example 2.

[0222] Physical properties of the Lessonia nigrescens sulfated fucanoligosaccharides 1-(1) to 1-(7)-2 of the present invention are shownbelow.

[0223] (a) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(1)

[0224] The results for the mass spectrometric analysis and assignment inNMR analyses are shown below, and the ¹H-NMR spectrum, ¹³C-NMR spectrum,and the mass spectrum of this oligosaccharide are shown in FIGS. 19, 20,and 21, respectively. In FIGS. 19 and 20, the vertical axes representthe signal intensity and the horizontal axes represent the chemicalshift value (ppm). In FIG. 21, the vertical axis represents the relativeintensity of the signals and the horizontal axis represents the m/zvalue.

[0225] Molecular weight; 718

[0226] MS m/z 216.5 [M-3Na⁺]³⁻, 336.0 [M-2Na⁺]²⁻, 695.0 [M−Na⁺]⁻

[0227] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Table 17.TABLE 17 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 90.8 5.41, d, 3.6 F1-2 75.14.38, dd, 3.6, 10.8 F1-3 75.1 4.11, dd, 10.8, 3.0 F1-4 81.0 4.84, d, 3.0F1-5 66.7 4.22, q, 6.0 F1-6 16.2 1.16, d, 6.0 F2-1 99.2 5.21, d, 3.0F2-2 76.4 4.29, m F2-3 67.8 4.29, m F2-4 81.5 4.55, d, 3.0 F2-5 67.84.31, q, 6.6 F2-6 16.3 1.16, d, 6.6

[0228] Component sugar: only L-fucose (2 molecules)

[0229] Sulfate residues: 4 molecules

[0230] Sodium: 4 molecules

[0231] The numbers for signal assignment in ¹H-NMR and

[0232]¹³C-NMR analyses are as indicated in formula (X) below:

[0233] (b) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(2)

[0234] The results for the mass spectrometric analysis and assignment inNMR analyses are shown below, and the ¹H-NMR spectrum, ¹³C-NMR spectrum,and the mass spectrum of the Lessonia nigrescens sulfated fucanoligosaccharide 1-(2) are shown in FIGS. 22, 23, and 24, respectively.In FIGS. 22 and 23, the vertical axes represent the signal intensity andthe horizontal axes represent the chemical shift value (ppm). In FIG.24, the vertical axis represents the relative intensity of the signalsand the horizontal axis represents the m/z value.

[0235] Molecular weight; 966

[0236] MS m/z 459.9[M-2Na⁺]²⁻, 942.9[M−Na⁺]⁻

[0237] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Table 18.TABLE 18 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 90.6 5.42, d, 3.6 F1-2 75.74.22, dd, 3.6, 10.4 F1-3 73.8 4.24, dd, 10.4, 2.4 F1-4 80.5 4.79, d, 2.4F1-5 66.9 4.24, q, 6.4 F1-6 16.3 1.18, d, 6.4 F2-1 98.3 5.32, d, 2.0F2-2 74.9 4.41, m F2-3 72.5 4.41, m F2-4 79.8 4.72, br-s F2-5 67.8 4.30,q, 6.5 F2-6 16.3 1.17, d, 6.5 F3-1 97.7 5.03, d, 3.9 F3-2 69.0 3.66, dd,3.9, 10.5 F3-3 69.3 3.93, dd, 10.5, 2.8 F3-4 81.4 4.51, d, 2.8 F3-5 66.94.46, q, 6.4 F3-6 16.3 1.19, d, 6.4

[0238] Component sugar: only L-fucose (3 molecules)

[0239] Sulfate residues: 5 molecules

[0240] Sodium: 5 molecules

[0241] The numbers for signal assignment in ¹H-NMR and ¹³C-NMR analysesare as indicated in formula (XI) below:

[0242] (c) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(3)

[0243] The results for the mass spectrometric analysis and assignment inNMR analyses are shown below, and the ¹H-NMR spectrum, ¹³C-NMR spectrum,and the mass spectrum are shown in FIGS. 25, 26, and 27, respectively.In FIGS. 25 and 26, the vertical axes represent the signal intensity andthe horizontal axes represent the chemical shift value (ppm). In FIG.27, the vertical axis represents the relative intensity of the signalsand the horizontal axis represents the m/z value.

[0244] Molecular weight; 1068

[0245] MS m/z 332.9 [M-3Na⁺]³⁻, 511.0 [M-2Na⁺]²⁻, 1045.0 [M-Na⁺]⁻

[0246] The results of ¹H-NMR and ¹³C-NMR analyses are shown in Table 19.TABLE 19 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, ¹³C-NMR coupling constant F1-1 90.3 5.42, d, 3.4 F1-2 75.54.39, dd, 3.4, 10.2 F1-3 74.6 4.22, dd, 10.2, 2.9 F1-4 78.0 4.80, d, 2.9F1-5 67.3 4.24, q, 6.6 F1-6 16.3 1.20, d, 6.6 F2-1 98.8 5.30, d, 3.3F2-2 75.0 4.41, m F2-3 75.0 4.30, m F2-4 80.7 4.81, d, 2.6 F2-5 68.44.25, q, 6.7 F2-6 16.2 1.18, d, 6.7 F3-1 98.5 5.24, d, 3.2 F3-2 75.74.31, dd, 3.2, 10.4 F3-3 66.5 4.26, dd, 10.4, 3.0 F3-4 81.7 4.57, d, 3.0F3-5 67.4 4.36, q, 6.6 F3-6 16.0 1.20, d, 6.6

[0247] Component sugar: only L-fucose (3 molecules)

[0248] Sulfate residues: 6 molecules

[0249] Sodium: 6 molecules

[0250] The numbers for signal assignment in ¹H-NMR and ¹³C-NMR analysesare as indicated in formula (XII) below:

[0251] (d) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(4)

[0252] By the structural analyses of the oligosaccharide as shown inExample 2, the structure of Lessonia nigrescens sulfated fucanoligosaccharide 1-(4) was determined to be the same structure as thesulfated fucan oligosaccharide 1-(2) shown in Example 2.

[0253] (e) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(5)

[0254] By the structural analyses of the oligosaccharide as shown inExample 2, the structure of Lessonia nigrescens sulfated fucanoligosaccharide 1-(5) was determined to be the same structure as thesulfated fucan oligosaccharide 1-(3) shown in Example 2.

[0255] (f) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(6)

[0256] The results of the mass spectrometric analysis for Lessonianigrescens sulfated fucan oligosaccharides 1-(6) of the presentinvention are shown below.

[0257] Molecular weight; 3461

[0258] MS m/z 361.5 [M-9Na⁺]⁹⁻, 409.62 [M-8Na⁺]⁸⁻, 471.42 [M-7Na⁺]⁷⁻,553.81 [M-6Na⁺]⁶⁻, 669.31 [M-5Na⁺]⁵⁻, 842.32 [M-4Na⁺]⁴⁻, 1130.83[M-3Na⁺]³⁻

[0259] Component sugar: only L-fucose (11 molecules)

[0260] Sulfate residues: 18 molecules

[0261] Sodium: 18 molecules

[0262] (g) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(7)-1

[0263] The results of the mass spectrometric analysis for Lessonianigrescens sulfated fucan oligosaccharide 1-(7)-1 of the presentinvention are shown below.

[0264] Molecular weight; 4659

[0265] MS m/z 442.82 [M-10Na⁺]¹⁰⁻, 494.72 [M-9Na⁺]⁹⁻, 559.32 [M-8Na⁺]⁸⁻,642.62 [M-7Na⁺]⁷⁻, 753.52 [M-6Na⁺]⁶⁻, 908.82 [M-5Na⁺]⁵⁻, 1141.43[M-4Na⁺]⁴⁻

[0266] Component sugar: only L-fucose (15 molecules)

[0267] Sulfate residues: 24 molecules

[0268] Sodium: 24 molecules

[0269] (h) Physical Properties of Lessonia nigrescens Sulfated FucanOligosaccharide 1-(7)-2

[0270] The results of the mass spectrometric analysis for Lessonianigrescens sulfated fucan oligosaccharide 1-(7)-2 of the presentinvention are shown below.

[0271] Molecular weight; 3564

[0272] MS m/z 373.12[M-9Na⁺]⁹⁻, 422.52 [M-8Na⁺]⁸⁻, 486.32 [M-7Na⁺]⁷⁻,571.01 [M-6Na⁺]⁶⁻, 689.61 [M-5Na⁺]⁵⁻, 868.02 [M-4Na⁺]⁴⁻, 1164.93[M-3Na⁺]³⁻

[0273] Component sugar: only L-fucose (11 molecules)

[0274] Sulfate residues: 19 molecules

[0275] Sodium: 19 molecules

Example 6 Preparation of Sulfated Fucan Oligosaccharide Using SulfatedFucan-Digesting Enzyme, and Purification and Structural Analysis thereof

[0276] (1) Preparation

[0277] 10 g of the fucoidan from cultured Kjellmaniella crassifolia asdescribed in Referential Example 1 was dissolved in 4.8 L of 17 mMimidazole-hydrochloride buffer (pH 7.5) containing 125 mM calciumchloride, 250 mM sodium chloride and 10% ethanol. 10 U of the sulfatedfucan-digesting enzyme was added thereto. The resulting mixture wasstirred at room temperature for 72 hours to obtain a Kjellmaniellacrassifolia fucoidan oligosaccharide solution. A small molecule fractionrecovered from the solution using an ultrafiltration device equippedwith hollow fibers with exclusion molecular weight of 10,000 wasdesignated as a sulfated fucan oligosaccharide fraction 3.

[0278] (2) Analysis

[0279] The sulfated fucan oligosaccharide fraction 3 obtained in (1)above was loaded onto a 1-L DEAE-Cellulofine A-800 column equilibratedwith 10 mM imidazole-hydrochloride buffer (pH 6.5) containing 100 mMsodium chloride. After adequately washing with the same buffer, elutionwas then carried out with a gradient of 100 mM to 1 M sodium chloride.The total sugar content and the total uronic acid content of each of theeluted fractions were measured according to the phenol-sulfuric acidmethod and the carbazole-sulfuric acid method, respectively. As aresult, the eluted fractions formed two peaks. The fractions in therespective peaks were subjected to mass spectrometric analyses.Determination of the saccharide compositions for the respective peaksshowed that they contained only fucose but did not contain uronic acid.Compositions of the oligosaccharides having the respective massesestimated based on the saccharide compositions are shown in Table 20.TABLE 20 Composition of oligosaccharide Molecular Sulfate Peak no.weight Fucose group Sodium 3-(1) 1564 5 8 8 1914 6 10 10 2016 6 11 112162 7 11 11 2264 7 12 12 2410 8 12 12 3-(2) 3110 10 16 16 3360 11 17 173462 11 18 18 3710 12 19 19 4308 14 22 22

[0280] It was shown that the sulfated fucan oligosaccharide fraction 3contained various sulfated fucan oligosaccharides as shown in the tableabove.

Example 7 Preparation of Sulfated Fucan Oligosaccharide Using SulfatedFucan-Digesting Enzyme, and Purification, Structural Analysis andPhysiological Activity Thereof

[0281] (1) In addition to the four distinct peaks observed inDEAE-Cellulofine column chromatography as described in Example 2-(2), abroad peak eluted with a higher salt concentration was observed. It wascollected, designated as an oligosaccharide 4 and subjected to NMRanalysis as described in Example 2. As a result, a spectrum almostidentical to that for the sulfated fucan oligosaccharide 1-(2) wasobserved. These results strongly suggested that the oligosaccharide 4had a structure in which several molecules of the oligosaccharide 1-(2)were connected each other. Then, the oligosaccharide 4 was digestedusing the sulfated fucan-digesting enzyme as described in Example 1, andthe digestion product was analyzed using HPLC. The majority of thereaction products was eluted at the same position as that for thesulfated fucan oligosaccharide 1-(2).

[0282] The molecular weight of the oligosaccharide 4 as determined bygel filtration using pullulan as a standard substance was shown to beabout triple of the oligosaccharide 1-(2).

[0283] Precise analysis of the ¹H-NMR spectrum for the oligosaccharide 4revealed that the repeat of seven saccharide residues was bound at the3-position of fucose F6 in formula (IV) via an α-(1→3) bond.

[0284] A monomer to a pentamer of the sulfated saccharide represented bygeneral formula (I) (i.e., sulfated saccharides of general formula (I)wherein n=1 to 5) were obtained from digestion products of a sulfatedpolysaccharide according to the method as described above.

[0285] wherein R is H or SO₃H and at least one of Rs is SO₃H.

[0286] As described above, it was confirmed that a sulfatedfucan-containing polysaccharide obtained from a brown alga such asKjellmaniella crassifolia was converted into smaller molecules bytreating it using a sulfated fucan-digesting enzyme, and that a sulfatedpolysaccharide containing a sulfated saccharide of the general formulaabove as an essential component of the constituting saccharide was thenobtained. The average molecular weight of the sulfated polysaccharideextracted at pH 6-8 at 95° C. for about 2 hour as determined by gelfiltration was about 200,000.

[0287] (2) The physiological activities of the sulfated saccharideobtained in (1) above were examined. As a result, an activity ofinducing HGF production as determined according to the method describedin Example 1-(2) in WO 00/62785 was observed for the sulfatedpolysaccharide of formula (I). In addition, the sulfated saccharides offormula (I) were shown to be very useful in retaining moisture asdetermined according to the method described in Examples 8 and 9 in WO99/41288.

[0288] The present invention provides a method for producing a sulfatedfucan oligosaccharide having a varying molecular weight, which is usefulas a reagent for glycotechnology, using a sulfated fucan-digestingenzyme. The present invention also provides various sulfated fucanoligosaccharides of which the structures have been determined.

1 1 1 1506 DNA Fucanobacter lyticus 1 agagtttgat cctggctcag attgaacgctggcggcaggc ttaacacatg caagtcgagc 60 ggaaacgaga atagcttgct attcggcgtcgagcggcgga cgggtgagta atgcttggga 120 atatgcctaa tggtggggga caacagttggaaacgactgc taataccgca taatgtctac 180 ggaccaaagg aggggattct tcggaacctttcgccatttg attagcccaa gtgagattag 240 ctagtaggta aggtaatggc ttacctaggcgacgatctct agctggtttg agaggatgat 300 cagccacact gggactgaga cacggcccagactcctacgg gaggcagcag tggggaatat 360 tgcacaatgg gcgaaagcct gatgcagccatgccgcgtgt gtgaagaagg ccttcgggtt 420 gtaaagcact ttcagcgagg aggaaagggtgtagattaat actctgcatc tgtgacgtta 480 ctcgcagaag aagcaccggc taacttcgtgccagcagccg cggtaatacg aggggtgcaa 540 gcgttaatcg gaattactgg gcgtaaagcgtgcgtaggtg gtttgttaag caagatgtga 600 aagccccggg ctcaacctgg gaactgcattttgaactggc aaactagagt tttgtagagg 660 gtagtggaat ttccagtgta gcggtgaaatgcgtagagat tggaaggaac atcagtggcg 720 aaggcggcta cctggacaga gactgacactgaggcacgaa agcgtgggga gcaaacagga 780 ttagataccc tggtagtcca cgccgtaaacgatgtcaact agccgtctgt agacttgatc 840 tgtgggtggc gtagctaacg cgctaagttgaccgcctggg gagtacggcc gcaaggttaa 900 aactcaaatg aattgacggg ggcccgcacaagcggtggag catgtggttt aattcgatgc 960 aacgcgaaga accttaccat cccttgacatcctactaagt tactagagat agtttcgtgc 1020 cttcgggaaa gtagtgacag gtgctgcatggctgtcgtca gctcgtgttg tgaaatgttg 1080 ggttaagtcc cgcaacgagc gcaacccctatccttatttg ctagcgcgta atggcgagaa 1140 ctctaaggag actgccggtg ataaaccggaggaaggtggg gacgacgtca agtcatcatg 1200 gcccttacgg gatgggctac acacgtgctacaatggcaag tacagagggc agcaataccg 1260 cgaggtggag cgaatcccac aaagcttgtcgtagtccgga ttggagtctg caactcgact 1320 ccatgaagtc ggaatcgcta gtaatcgtagatcagaatgc tacggtgaat acgttcccgg 1380 gccttgtaca caccgcccgt cacaccatgggagtgggttg caaaagaagt ggctagttta 1440 acccttcggg gaggacggtc accactttgtgattcatgac tggggtgaag tcgtaacaag 1500 gtagcc 1506

What is claimed is:
 1. A sulfated fucan oligosaccharide obtainable bythe method comprising: allowing a sulfated fucan-digesting enzymederived from Alteromonas sp. SN-1009 to act on the substance selectedfrom the group consisting of: a sulfated fucan having the followingchemical and physical properties: (1) containing fucose as aconstituting saccharide; (2) containing a sulfated saccharide of generalformula (I) as an essential component of the constituting saccharide:

wherein R is H or SO₃H, at least one of R's is SO₃H and n is an integerof 1 or more; and (3) being converted into smaller molecules by asulfated fucan-digesting enzyme derived from Alteromonas sp. SN-1009 togenerate at least one compound of general formulas (II), (III), (XIII),(XIV), (XV) and (XVI):

wherein R is H or SO₃H, at least one of R's is SO₃H in all formulasabove, and a sulfated fucan oligosaccharide of general formula (I):

wherein R is H or SO₃H, at least one of R's is SO₃H and n is 1 to 5; andcollecting a digestion product.
 2. A degradation product of sulfatedfucan obtainable by the method comprising: allowing a sulfatefucan-digesting enzyme derived from Alteromonas sp. SN-1009 to act onthe substance selected from the group consisting of: a sulfated fucanhaving the following chemical and physical properties: (1) containingfucose as a constituting saccharide; (2) containing a sulfatedsaccharide of general formula (I) as an essential component of theconstituting saccharide:

wherein R is H or SO₃H, at least one of R's is SO₃H and n is an integerof 1 or more; and (3) being converted into smaller molecules by asulfated fucan-digesting enzyme derived from Alteromonas sp. SN-1009 togenerate at least one compound of general formulas (II), (III), (XIII),(XIX), (XV) and (XVI):

wherein R is H or SO₃H, at least one of R's is SO₃H in all formulasabove; and a sulfated fucan oligosaccharide of general formula (I):

wherein R is H or SO₃H, at lease one of R's is SO₃H and n is 1 to 5.